Functional Analysis of Genes and Genomic Variants in Animal Models

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PROJECT 2 ABSTRACT/RESEARCH SUMMARY. Project 1 of DGAP takes advantage of a robust clinical referral network and innovations in genome analysis approaches to identify disease associated genes-of-interest using BCAs. Akin to other human genomics research projects (e.g., GWAS, WES/WGS), we are generating a rapidly expanding candidate gene list that is outpacing the functional analysis to validate the causal relationship and understand the underlying pathophysiology. Use of an animal model to disrupt the human ortholog and recapitulate the clinical phenotype remains the best functional evidence for gene function and pathogenicity of gene variants. Major advances in gene targeting methods now make it possible to functionally annotate human candidate genes in a higher throughput and precisely targeted fashion. Further, the biologic advantages and wealth of transgenic reporters allows for rapid phenotypic analysis from direct visualization of the biological process of interest. The goal of Project 2 is to use the convergence of advanced gene editing state-of-the-art biological system analyses and convergent bioinformatics to establish rapidly functional evidence for genes-of-interest. We have designed Project 2 to provide ready-to-use deliverables to the scientific community. Aim 1 utilizes high-throughput in vivo spatiotemporal expression analysis and morpholino-mediated gene knockdown to rapidly analyze the function of candidate genes (approximately 30 per year) discovered at the BCA breakpoints. Aim 2 applies high-throughput targeted mutagenesis using CRISPR/Cas9 approach to generate loss-of-function zebrafish models (approximately 10-15 genes per year) to provide functional evidence for consequences of DGAP gene disruptions in the appropriate biologic context. All organ systems will be examined via rapid phenotype assessment strategy, supported by a consultant team of zebrafish biologists as needed, while genes with potential function in neuro-development will be forwarded for analysis in Project 3. Aim 3 takes advantage of the animal models to carry out high-throughput analysis of human gene variants to determine pathogenicity of the mutation (approximately 10-12 variants per year). Project 2 will make a significant contribution to functional gene annotation discovered through the DGAP pipeline, innovate strategies to analyze human genome variants, and generate deliverables to the scientific community to catalyze biological explorations across organ systems.